SUMMARY (AIM 2): SYSTEMS PHARMACOLOGY CORE The Systems Pharmacology Core will provide all investigators and trainees who are part of this proposal, as well as individuals selected for funded internal research projects, access to an integrated resource for deep molecular profiling of cells and tissues and for analyzing the resulting data using a diversity of computational tools. The core draws on the resources of the Laboratory of Systems Pharmacology (LSP), which co-localizes postdocs and students from multiple research groups with professional PhD-level research staff who lead technology platforms. This will result in close and regular interaction between research projects and diverse measurement technologies both as a means to ensure continuous improvement in omic technologies as well as to assist investigators and trainees with experimental design and interpretation. Staff involved in the Systems Pharmacology Core also have a role in education and training as described under education and outreach in Aim 3. The LSP embraces a philosophy of continuous process improvement. For example, in the last year we implemented, and published in high impact journals, new approaches to single cell sequencing, focused proteomics and highly multiplexed single-cell imaging. To promote innovation, staff scientists charged with individual technologies will attend conferences and workshops on the latest technologies and continue to publish their work as primary and contributing authors. Aim 2.1 will support next generation sequencing and RNA-Seq using three complementary technologies that differ in cost, degree of coverage, throughput of samples and use of bulk vs. single cells. Aim 2.2 will provide mass spectrometry-based shotgun proteomics using isobaric tags optimized in the MS3 mode as well as focused proteomics using the recently developed TOMAHAQ method (in lieu of multiple reaction monitoring), and phospho-mass spectrometry using the latest generation Orbitrap Fusion? Lumos? Tribrid? Mass Spectrometer. Absolute quantification of peptides will aid in data interpretation and data fusion. Aim 2.3 will enable highly multiplexed (20-40 channel) imaging of cultured cells before and after exposure to drugs using an innovative Cyclic Immunofluorescence (CycIF) method developed at the LSP. Aim 2.4 will support metabolomic profiling of cell and serum samples using a targeted method that measures the levels of ~300 metabolites of known identity based on a library of reference compounds. Aim 2.5 will perform multiplex immunoassays on cytokines and growth factors as well as high dimensional flow cytometry and CyTOF mass cytometry to deeply profile human blood. Aim 2.6 will provide access to training and core technologies for general purpose data analysis (clustering, regression, dimensionality reduction) as a complement to specific modeling efforts in individual research projects.